Retroviral vectors are relevant for a range of applications, including gene therapy. However, progress in lentiviral gene therapy, for example, has been hampered by the requirement for production of purified lentiviral vectors with high titre.
A major limitation in the production of lentiviral vectors is the lack of appropriate stable producer cells and the low viral titre produced by most packaging cell lines. Currently, all large scale lentiviral production is generated by transient transfection methods. Until the problem of stable packaging cell lines is solved, lentiviral production cannot be fully industrialized.
The main challenges with making stable producer cell lines include the fact that high numbers of transfer vector transgenes are needed to generate appropriate viral titres. Transfer vector transgenes comprise the gene of interest which is to be delivered by the viral vector and the elements required for integration of the gene of interest into the host genome. The number of RNA transgenes which can be accumulated in the cytoplasm of producer cells is limited because the long transcripts of the transgenes are poorly transcribed and poorly exported from the nucleus.
In addition, high levels of lentiviral proteins such as gagpol and envelope proteins are needed to produce required lentiviral titres. This is difficult to achieve because gagpol is unstable and difficult to express and fold correctly. Another problem is the basal toxicity associated with the protease activity of gagpol. Further, a number of preferred envelopes for lentiviral pseudotyping—such as VSV-G—cause syncytial formation and are also basally toxic.
Accordingly, there is a need for alternative producer cells and methods for producing retroviral vectors.